Therapy for promoting cell growth

ABSTRACT

There is disclosed the use of a composition for promoting neuronal growth of neurons in tissues of the central or peripheral nervous system. There is also disclosed a method for inducing proliferation or differentiation of neuronal cells.

TECHNICAL FIELD

The present invention generally relates to methods and compositions forpromoting neuronal outgrowth and proliferation. The present inventionalso relates to an in vitro method for promoting cell growth.

BACKGROUND

Nervous system injuries affect numerous people every year. As a resultof this high incidence of neurological injuries, nerve regeneration andrepair is becoming a rapidly growing field dedicated to the discovery ofnew ways to recover nerve functionality after injury. However up to now,clinically repairing central nervous system (CNS) lesions and recoveringneurological functions for patients suffering from nervous systeminjuries have been problematic. Thus, patients with various forms ofnervous system diseases, such as amyotrophic lateral sclerosis (ALS) andsenile dementia, have always been told by their physicians that it wouldbe difficult to recover their neurological functions.

The ability of neurons to extend neurites (such as axons and dendrites)is of prime importance in establishing neuronal connections duringdevelopment. It is also required during neuroregeneration tore-establish connections destroyed as a result of a lesion. However,axons in the central nervous system have a very limited capacity tore-grow after a lesion. Thus, for diseases such as senile dementia, inwhich there is a progressive degeneration of neuronal cells, theresearch of therapeutic agents or molecules which are able to stimulateneuronal cell outgrowth and proliferation, will open a new therapeuticstrategy which focuses on neural repair and restoring neurologicalfunction.

There is a need to improve methods of treating nervous system injuriesand neurological diseases. More specifically, there is a need to provideregenerative therapies which can promote neuronal outgrowth andproliferation of neurons so as to enable damaged or diseases nerves tofunction again.

SUMMARY

According to a first aspect, there is provided the use of a compositioncomprising at least two components selected from the group consisting ofthe following: Radix et Rhizoma Salviae Miltiorrhizae (Red Sage root orDan Shen), Prunus Persica (Peach seed or Taoren), Radix Polygalae (rootof thinleaf milkwort, Polygala tenuifolia Willd., Polygala sibirica L.or Yuanzhi) and Rhizoma acori Tatarinowii (rhizome of grassleafsweetflag or Shichangpu) and combinations thereof, and an optionalcomponent selected from the group consisting of: Radix Astragali (rootof Membranous Milkvetch or Huang Qi), Radix Paeoniae Rubra (Red Peonyroot, Paeonia lactiflora Pall, Paeonia veitchii Lynch or Chi Shao),rhizome of Ligusticum Chuanxiong (Chuan Xiong), Flower of CarthamusTinctorius (Safflower or HongHua), and radix angelicae sinensis (root ofChinese Angelica or DanGui) and combinations thereof, in the manufactureof a medicament for promoting neuronal outgrowth and proliferation ofneurons in tissues of the central or peripheral nervous system.

In one embodiment, the composition of the first aspect further comprisesan optional component selected from the group consisting of: Buthusmartensii (dried body of scorpio or Quanxie), Eupolyphaga SeuSeteleophaga (dried body of ground beetle, Eupolyphaga sinensis Walker,Steleophaga plancyi or Tubiechong), Calculus Bovis Artifactus (Naturalor Artificial cow-bezoar or Rengong Niuhuang), Cornu Saigae Tataricae(Antelope Horn or Lingyangjiao) and dried body of leeches (Hirudo,Whitmania pigra Whitman, Hirudo nipponica Whitman Whitmania acranulataWhitman or Shuizhi).

Advantageously, the medicament can induce neuronal outgrowth of neuronsand proliferation in diseased or injured tissue where significant tissueshrinkage, loss, atrophy or cell death has occurred. Promoting neuronaloutgrowth and proliferation of neurons enable damaged or disease nerveto perform directional outward growth that may introduce beneficialsynapse connection or be replaced with new functioning cells. Thecomposition may also promote stem cell differentiation and recruitmentinto the diseased or injured tissue.

In one embodiment, there is provided the use of a composition comprisinga first component selected from the group consisting of the followingcomponents: Radix et Rhizoma Salviae Miltiorrhizae (Red Sage root or DanShen), Prunus Persica (Peach seed or Taoren), Radix Polygalae (root ofthinleaf milkwort, Polygala tenuifolia Willd., Polygala sibirica L. orYuanzhi), Buthus martensii (dried body of scorpio or Quanxie), Rhizomaacori Tatarinowii (rhizome of grassleaf sweetflag or Shichangpu),Eupolyphaga Seu Seteleophaga (dried body of ground beetle, Eupolyphagasinensis Walker, Steleophaga plancyi or Tubiechong), Calculus BovisArtifactus (Natural or Artificial cow-bezoar or Rengong Niuhuang), andCornu Saigae Tataricae (Antelope Horn or Lingyangjiao) and combinationsthereof, and an optional second component selected from the groupconsisting of: Radix Astragali (root of Membranous Milkvetch or HuangQi), Radix Paeoniae Rubra (Red Peony root, Paeonia lactiflora Pall,Paeonia veitchii Lynch or Chi Shao), rhizome of Ligusticum Chuanxiong(Chuan Xiong), Flower of Carthamus Tinctorius (Safflower or HongHua),radix angelicae sinensis (root of Chinese Angelica or DanGui) and driedbody of leeches (Hirudo, Whitmania pigra Whitman, Hirudo nipponicaWhitman Whitmania acranulata Whitman or Shuizhi) and combinationsthereof, in the manufacture of a medicament for treating patients havinga condition selected from the group of depression, psychiatricindications, natural aging and traumatic brain cell death, fordiminishing the effects of stroke or neurodegeneration in predisposedsubjects or subjects at risk of stroke or neurodegeneration, fortreating a patient having a diseased or injured tissues of the centraland peripheral nervous system and for promoting cell growth.

According to a second aspect, there is provided the use of a compositioncomprising at least two of the following components: Radix Astragali(root of Membranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or HongHua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii(rhizome of grassleaf sweetflag or Shichangpu) in the manufacture of amedicament for treating patients having a condition selected from thegroup of depression, psychiatric indications, natural aging andtraumatic brain cell death.

Whilst not bound by theory, we believe that the medicament as disclosedherein may be useful for treating patients with depression or otherpsychiatric indications such as schizophrenia and anxiety disorders bypromoting the regulation of hormonal balance that may have beendisrupted by loss of cell function.

According to a third aspect, there is provided the use of a compositioncomprising at least three of the following components: Radix Astragali(root of Membranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or HongHua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii(rhizome of grassleaf sweetflag or Shichangpu) in the manufacture of amedicament for diminishing the effects of stroke or neurodegeneration inpredisposed subjects or subjects at risk of stroke or neurodegeneration.

According to a fourth aspect, there is provided the use of a compositioncomprising at least three of the following components: Radix Astragali(root of Membranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or HongHua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii(rhizome of grassleaf sweetflag or Shichangpu) in the manufacture of amedicament for promoting cell growth.

In one embodiment, the medicament may promote controlled growth ofchondrocytes, skeletal muscle cells, myocardiums, smooth muscle cells,hepatocytes, kidney cells and epithelial skin cells. A medicament thatpromotes cell growth may be used for treating conditions such asrheumatoid arthritis, muscle degenerative disorders, stroke, kidney andliver diseases. The medicament may also be used for delaying agingprocesses by improving epithelial or epidermal cell proliferations orfunctions.

According to a fifth aspect, there is provided the use of at least oneof the following components: Radix Astragali (root of MembranousMilkvetch or Huang Qi), Radix et Rhizoma Salviae Miltiorrhizae (Red Sageroot or Dan Shen), Radix Paeoniae Rubra (Red Peony root, Paeonialactiflora Pall, Paeonia veitchii Lynch or Chi Shao), rhizome ofLigusticum Chuanxiong (Chuan Xiong), radix angelicae sinensis (root ofChinese Angelica or DanGui), Flower of Carthamus Tinctorius (Saffloweror HongHua), Prunus Persica (Peach seed or Taoren), Radix Polygalae(root of thinleaf milkwort, Polygala tenuifolia Willd., Polygalasibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii (rhizome ofgrassleaf sweetflag or Shichangpu) in the manufacture of a medicament,and a supplemental agent, for treating a patient having a diseased orinjured tissues of the central or peripheral nervous system.

In one embodiment, there is provided the use of the composition asdefined above, wherein the composition comprises at least 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13 or 14, of the components. The supplemental agentmay be mineral supplements, vitamins, herbal supplements, WesternMedicine or fish oils.

According to a sixth aspect, there is provided a cell culture reagentfor promoting cell survival and growth comprising a culture medium andat least one of the following components: Radix Astragali (root ofMembranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or HongHua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii(rhizome of grassleaf sweetflag or Shichangpu).

In one embodiment, there is provided a cell culture reagent comprisingthe composition as defined in the sixth aspect, wherein the compositioncomprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of thecomponents.

In another embodiment, the cell culture reagent may promote cellsurvival and growth of neurons, stem cells, chondrocytes, skeletalmuscle cells, myocardiums, smooth muscle cells, hepatocytes, kidneycells, islets of langerhans and epithelial skin cells.

According to a seventh aspect, there is provided a method for promotingcell survival, inducing proliferation or differentiation of cells,comprising contacting said cells with an effective amount of at leastthree of the following components: Radix Astragali (root of MembranousMilkvetch or Huang Qi), Radix et Rhizoma Salviae Miltiorrhizae (Red Sageroot or Dan Shen), Radix Paeoniae Rubra (Red Peony root, Paeonialactiflora Pall, Paeonia veitchii Lynch or Chi Shao), rhizome ofLigusticum Chuanxiong (Chuan Xiong), radix angelicae sinensis (root ofChinese Angelica or DanGui), Flower of Carthamus Tinctorius (Saffloweror HongHua), Prunus Persica (Peach seed or Taoren), Radix Polygalae(root of thinleaf milkwort, Polygala tenuifolia Willd., Polygalasibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii (rhizome ofgrassleaf sweetflag or Shichangpu).

The contacting step of the seventh aspect may be undertaken in vitro. Anin vitro contacting step may promote cell growth, differentiation orcell survival would facilitate in vitro culture of various cells thatmay be used for tissue engineering or ex vivo therapeutic uses. In oneembodiment, the effective amount is 1 g to 8 g. In another embodiment,the neuronal cells are vertebrate neuronal cells, preferably humanneuronal cells. In one embodiment, the neuronal cells are derived fromcortical neuronal cells.

According to an eight aspect, there is provided a method of promotingneuronal outgrowth and proliferation of neurons in tissues of thecentral or peripheral nervous system of a patient comprising the step ofadministering to said patient a composition comprising at least twocomponents selected from the group consisting of the following: Radix etRhizoma Salviae Miltiorrhizae (Red Sage root or Dan Shen), PrunusPersica (Peach seed or Taoren), Radix Polygalae (root of thinleafmilkwort, Polygala tenuifolia Willd., Polygala sibirica L. or Yuanzhi)and Rhizoma acori Tatarinowii (rhizome of grassleaf sweetflag orShichangpu) and combinations thereof.

According to a ninth aspect, there is provided a method of treatingpatients having a condition selected from the group of depression,psychiatric indications, natural aging and traumatic brain cell deathcomprising the step of administering to said patients at least two ofthe following components: Radix Astragali (root of Membranous Milkvetchor Huang Qi), Radix et Rhizoma Salviae Miltiorrhizae (Red Sage root orDan Shen), Radix Paeoniae Rubra (Red Peony root, Paeonia lactifloraPall, Paeonia veitchii Lynch or Chi Shao), rhizome of LigusticumChuanxiong (Chuan Xiong), radix angelicae sinensis (root of ChineseAngelica or DanGui), Flower of Carthamus Tinctorius (Safflower orHongHua), Prunus Persica (Peach seed or Taoren), Radix Polygalae (rootof thinleaf milkwort, Polygala tenuifolia Willd., Polygala sibirica L.or Yuanzhi) and Rhizoma acori Tatarinowii (rhizome of grassleafsweetflag or Shichangpu).

According to a tenth aspect, there is provided a method for diminishingthe effects of stroke or neurodegeneration in predisposed subjects orsubjects at risk of stroke or neurodegeneration comprising the step ofadministering to said subjects at least three of the followingcomponents: Radix Astragali (root of Membranous Milkvetch or Huang Qi),Radix et Rhizoma Salviae Miltiorrhizae (Red Sage root or Dan Shen),Radix Paeoniae Rubra (Red Peony root, Paeonia lactiflora Pall, Paeoniaveitchii Lynch or Chi Shao), rhizome of Ligusticum Chuanxiong (ChuanXiong), radix angelicae sinensis (root of Chinese Angelica or DanGui),Flower of Carthamus Tinctorius (Safflower or Hong Hua), Prunus Persica(Peach seed or Taoren), Radix Polygalae (root of thinleaf milkwort,Polygala tenuifolia Willd., Polygala sibirica L. or Yuanzhi) and Rhizomaacori Tatarinowii (rhizome of grassleaf sweetflag or Shichangpu).

Glossary of Terms

This section is intended to provide guidance on the interpretation ofthe words and phrases set forth below (and where appropriate grammaticalvariants thereof).

The term “neuronal outgrowth” in the specification relates to thegeneral directional outward growth of axons and dendrites. Neuronaloutgrowth is important in synapse formation or development.

Unless specified otherwise, the terms “comprising” and “comprise”, andgrammatical variants thereof, are intended to represent “open” or“inclusive” language such that they include recited elements but alsopermit inclusion of additional, unrecited elements.

As used herein, the term “about” as used in relation to a numericalvalue means, for example, +50% or +30% of the numerical value,preferably +20%, more preferably +10%, more preferably still +5%, andmost preferably +1%. Where necessary, the word “about” may be omittedfrom the definition of the invention.

The term “treatment” includes any and all uses which remedy a diseasestate or symptoms, prevent the establishment of disease, or otherwiseprevent, hinder, retard, or reverse the progression of disease or otherundesirable symptoms in any way whatsoever. Hence, “treatment” includesprophylactic and therapeutic treatment.

Throughout this disclosure, certain embodiments may be disclosed in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosed ranges.Accordingly, the description of a range should be considered to havespecifically disclosed all the possible sub-ranges as well as individualnumerical values within that range. For example, description of a rangesuch as from 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

DETAILED DESCRIPTION

The present invention provides the use of a composition that comprisesat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of thefollowing components: Radix et Rhizoma Salviae Miltiorrhizae (Red Sageroot or Dan Shen), Prunus Persica (Peach seed or Taoren), RadixPolygalae (root of thinleaf milkwort, Polygala tenuifolia Willd.,Polygala sibirica L. or Yuanzhi), Buthus martensii (dried body ofscorpio or Quanxie), Rhizoma acori Tatarinowii (rhizome of grassleafsweetflag or Shichangpu), Eupolyphaga Seu Seteleophaga (dried body ofground beetle, Eupolyphaga sinensis Walker, Steleophaga plancyi orTubiechong), Calculus Bovis Artifactus (Natural or Artificial cow-bezoaror Rengong Niuhuang), and Cornu Saigae Tataricae (Antelope Horn orLingyangjiao) and combinations thereof, in the manufacture of amedicament for promoting neuronal outgrowth and proliferation of neuronsor stem cells in tissues of the central or peripheral nervous system.

In one embodiment, the composition further comprises a second componentselected from the group consisting of: Radix Astragali (root ofMembranous Milkvetch or Huang Qi), Radix Paeoniae Rubra (Red Peony root,Paeonia lactiflora Pall, Paeonia veitchii Lynch or Chi Shao), rhizome ofLigusticum Chuanxiong (Chuan Xiong), Flower of Carthamus Tinctorius(Safflower or HongHua), radix angelicae sinensis (root of ChineseAngelica or DanGui) and dried body of leeches (Hirudo, Whitmania pigraWhitman, Hirudo nipponica Whitman Whitmania acranulata Whitman orShuizhi) and combinations thereof.

The medicament promotes neuronal outgrowth and proliferation of neuronsor stem cells in injured or diseased tissues which may occur patientswith any of the following diseases: amyotrophic lateral sclerosis (ALS),brain abscess, brain ischemia, brain atrophy associated with diabetes,cerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL), cerebrovascular disorders, corticobasalganglionic degeneration (CBGD), chronic ischemia, Creutzfeldt-JakobSyndrome, Dandy-Walker Syndrome, Duchenne Muscular Dystrophy, seniledementia, dementia associated with Acquired Immunodeficiency Syndrome(AIDS), encephalomyelitis, essential tremor, friedreich ataxia,gerstmann straussler-scheinker disease, Huntington disease,hydrocephalus, hypoxia, fatal familial insomnia, transient ischemicattack, kuru, Landau-Kleffner Syndrome, Lewy body disease,Machado-Joseph disease, bacterial and viral meningitis, migrainedisorders, myelitis, olivopotocerebellar atrophies, pantothenatekinase-associated neurodegeneration, poliomyelitis, postpoliomyelitissyndrome, prion diseases, pseudotumor cerebri, shy-drager syndrome,infantile spasms, progressive supranuclear palsy, Steinert disease,syringomyelia, thalamic diseases, Tic disorders, Tourette syndrome andUveomeningoencephalitic syndrome.

The composition can also be used to treat patients having a conditionselected from the group of psychiatric indications such as anxietydisorders, schizophrenia, depression and post-natal depression, naturalaging, traumatic brain cell death and other neurologic manifestationssuch as amnesia, back pain, vertigo, unconsciousness, phantom limb,olfaction disorders, neck pain, headache, migraines, spasm and speechdisorders.

In another embodiment, the composition may diminish the effect of strokeor neurodegeneration from predisposed subjects or subjects at risk ofstroke or neurodegerenation. The neurodegeneration may be caused bydiseases selected from the group of Alzheimer's disease, amyotrophiclateral sclerosis (ALS), brain abscess, brain ischemia, brain atrophyassociated with diabetes, cerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL), cerebrovasculardisorders, corticobasal ganglionic degeneration (CBGD), chronicischemia, Creutzfeldt-Jakob Syndrome, Dandy-Walker Syndrome, DuchenneMuscular Dystrophy, senile dementia, dementia associated with AcquiredImmunodeficiency Syndrome (AIDS), encephalomyelitis, essential tremor,friedreich ataxia, gerstmann straussler-scheinker disease, Huntingtondisease, hydrocephalus, hypoxia, fatal familial insomnia, transientischemic attack, kuru, Landau-Kleffner Syndrome, Lewy body disease,Machado-Joseph disease, bacterial and viral meningitis, migrainedisorders, myelitis, olivopotocerebellar atrophies, pantothenatekinase-associated neurodegeneration, Parkinson's disease, poliomyelitis,postpoliomyelitis syndrome, prion diseases, pseudotumor cerebri,shy-drager syndrome, Steinert disease, infantile spasms, progressivesupranuclear palsy, syringomyelia, thalamic diseases, Tic disorders,Tourette syndrome, Uveomeningoencephalitic syndrome, global and focalischemia and other cardiovascular diseases, in predisposed subjects.

It is envisaged that NeuroAid™ or a NeuroAid™-like composition (e.g. acomposition described below), optionally in combination with availablesupplemental agents that may be useful for the general well being ofneurons or for the treatment of various nervous system diseases andother degenerative diseases. The supplemental agents may be vitamins,mineral supplements, herbal supplements, fish oils and Western medicine.Typically, the supplemental agent used in combination with NeuroAid™ ora NeuroAid™-like composition is one that targets a different mechanismfrom NeuroAid™ or a NeuroAid™-like composition. For example, the Westernmedicine may be growth factors typically used in improving recoverypotential in the patients with degenerative diseases. Examples ofsuitable agents used in Western medicine include Calcium ChannelBlockers (D-600, Diltiazem, Nitrendipine, Nimodipine, Nifedipine,Flunarizine, Fluspirilene, Isradipine, Nicardipine, PY 108-068,Verapamil and Triapamil), Calcium chelator (DP-b99), free radicalscavengers (Ebselen, Tirilazad, NXY-059), GABA receptor agonists(Diazepam, Baclofen), AMPA agonists (ZK 200775/MPQX), competitive NMDAantagonists (aptiganel/cerestat, CP 101,606, dextrophan, MK801/dizocilpine, remacemide), glycine site antagonists (GV 150526, ACEA1021), polyamine site antagonists (eliprodil), growth factors (bFGF),Sodium channel blockers (fosphenytoin, 619C89), potassium channel opener(BMS 204352, cromakalim, levcromakalim, aprikalim, pinacidil, diazoxide,nicorandil, minoxidil), piracetam, adenosine transport inhibitor(propentofylline), gangliosides GM (non NMDA antagonist), presynapticglutamate release inhibitors, clazosentan, desmoteplase, viprinex(ancrod), tenecteplase (TNKase; Metalyse), alteplase, cyclic nitrones,TWEAK (TNF-like weak inducer of apoptosis) receptor, thrombolyticatreatments (urokinase, streptokinase, t-PA/tissue plasminogen activatoror recombinant urokinase), anistreplase, riluzole, and disufenton sodium(NXY 059), candesartan, AX-200 (G-CSF, Fligrastim), caffeinol(caffeine+ethanol), enecadin, microplasmin, sonolysis+tPA, V-10153,HTUPA, solulin, piclozotan, S-0139, S-18986, AEOL-10150, AL-208,KN-38-7271, phridoxal 5-phosphate, Neu-2000KL, ONO-2231, PGX-100,RVX-208, SUN-N4057, SUN-N8075, TAT-NR2B9c, GLP-1-expressing stem celltherapy, Msc-1 (SA-4503, AGY-94806)), NH-02D, S-0139 259, tissueprotective cytokines (Lu-AA24493), V10153 270 (BB-10153, TAPgen),combined use of statins and other cholesterol lowering drugs,erythropoietin, cerebrolysin and CDP-choline(cytidine-5′-diphosphocholine).

There is provided a method for inducing proliferation or differentiationof cells, comprising contacting said cells with an effective amount ofat least one of the following components: Radix Astragali (root ofMembranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or HongHua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi), Buthus martensii (dried bodyof scorpio or Quanxie), Rhizoma acori Tatarinowii (rhizome of grassleafsweetflag or Shichangpu), dried body of leeches (Hirudo, Whitmania pigraWhitman, Hirudo nipponica Whitman, Whitmania acranulata Whitman orShuizhi), Eupolyphaga Seu Seteleophaga (dried body of ground beetle,Eupolyphaga sinensis Walker, Steleophaga plancyi or Tubiechong),Calculus Bovis Artifactus (Natural or Artificial cow-bezoar or RengongNiuhuang), and Cornu Saigae Tataricae (Antelope Horn or Lingyangjiao).

The composition may promote cell growth or cell survival wouldfacilitate in vitro culture of various cells that may be used for tissueengineering or ex vivo therapeutic uses. In one embodiment, the patientreceiving the cultured cells is also to be administered with NeuroAid™.

NeuroAid™ and Similar Compositions

The ingredients set forth above may be present in the composition in arelatively crude form (e.g. unprocessed or crushed herbs) or in a morerefined form (e.g. purified extracts).

In one embodiment, NeuroAid™ from Moleac Pte Ltd is used. NeuroAid™ is aTCM product in capsule form comprising 9 herbal components and 5 animalcomponents. NeuroAid™ comprises Radix Astragali (root of MembranousMilkvetch or Huang Qi), Radix et Rhizoma Salviae Miltiorrhizae (Red Sageroot or Dan Shen), Radix Paeoniae Rubra (Red Peony root, Paeonialactiflora Pall, Paeonia veitchii Lynch or Chi Shao), rhizome ofLigusticum Chuanxiong (Chuan Xiong), radix angelicae sinensis (root ofChinese Angelica or DanGui), Flower of Carthamus Tinctorius (Saffloweror HongHua), Prunus Persica (Peach seed or Taoren), Radix Polygalae(root of thinleaf milkwort, Polygala tenuifolia Willd., Polygalasibirica L. or Yuanzhi), Rhizoma acori Tatarinowii (rhizome of grassleafsweetflag or Shichangpu), Buthus martensii (dried body of scorpio orQuanxie), dried body of leeches (Hirudo, Whitmania pigra Whitman, Hirudonipponica Whitman, Whitmania acranulata Whitman or Shuizhi), EupolyphagaSeu Seteleophaga (dried body of ground beetle, Eupolyphaga sinensisWalker, Steleophaga plancyi or Tubiechong), Calculus Bovis Artifactus(Natural or Artificial cow-bezoar or Rengong Niuhuang), and Cornu SaigaeTataricae (Antelope Horn or Lingyangjiao).

In another embodiment, NeuroAid II comprises Radix Astragali (root ofMembranous Milkvetch or Huang Qi), Radix et Rhizoma SalviaeMiltiorrhizae (Red Sage root or Dan Shen), Radix Paeoniae Rubra (RedPeony root, Paeonia lactiflora Pall, Paeonia veitchii Lynch or ChiShao), rhizome of Ligusticum Chuanxiong (Chuan Xiong), radix angelicaesinensis (root of Chinese Angelica or DanGui), Flower of CarthamusTinctorius (Safflower or Hong Hua), Prunus Persica (Peach seed orTaoren), Radix Polygalae (root of thinleaf milkwort, Polygala tenuifoliaWilld., Polygala sibirica L. or Yuanzhi) and Rhizoma acori Tatarinowii(rhizome of grassleaf sweetflag or Shichangpu). NeuroAid™, which may beregistered under different names in different countries (e.g. in SouthAfrica it is marketed as Strocaid™ or Danqi Piantan Jiaonang™) ismanufactured by and available commercially in the People's Republic ofChina from Tianjin Shitian Pharmaceutical Group Co., Ltd (located in theJianxin Industrial area, Wangwenzhuang town, Xiqing district, TianjinCity, China; Postal Code 300381). It is also available from Moleac PteLtd (formerly Molecular Acupuncture Pte Ltd), the main licensee outsideof the People's Republic of China (11 Biopolis Way, Helios #09-08Singapore 138667).

For the avoidance of doubt, NeuroAid™ not only includes NeuroAid™ in theform in which it is currently marketed but also includes futureformulations of NeuroAid™ which may, for example, be marketed by TianjinShitian Pharmaceutical Group Co., Ltd or Moleac Pte Ltd. Such futureformulations may, for example, vary in dosage amounts or theconcentration of its active ingredients etc. NeuroAid™ is also known asMLC 601 and the terms “NeuroAid™” and “MLC 601” can be usedinterchangeably. Similarly NeuroAid II is also known as MLC 901 and theterms “NeuroAid II” and “MLC 901” can be used interchangeably.

Neuroprotectants

Using various mechanisms, neuroprotectants are compounds that preserveneuronal tissue at risk of dying during the course of diseases thatadversely cause neurodegeneration. Some neuroprotectant agents includeantioxidants (e.g. selenium, 30 vitamin E, vitamin C, glutathione,cysteine, flavinoids, quinolines, enzymes with; reducing activity, etc),N-methyl-D-aspartate Receptor Antagonists (Dextrorphan, Selfotel,Magnesium), Narcotic Receptor antagonist (Nalmefene (Cervene),Ca-channel blockers, Na-channel modulators (Lubeluzole),Alpha-aminobutyric acid agonist (Clomethiazole), glutamate receptormodulators, serotonin receptor agonists (repinotan), phospholipids,free-radical scavenger (Tirilazad, and NXY-059), astrocyte activationinhibitor (ONO 2506), monoclonal antibodies such as anti-ICAM-1(Enlimomab), Human anti-leukocytic antibody, Hu23F2G, membranestabilization agent CDP-choline (Citicholine), Fibroblast growth factor(Fiblast), unsaturated- and polyunsaturated fatty acids, estrogens andselective estrogen receptor modulators (SEAMS), progestins, thyroidhormone and thyroid hormone-mimicking compounds, cyclosporin A andderivatives, thalidomide and derivatives, methylxanthines,Mono-Amine-Oxydase inhibitors (IMAO), serotonin-, noradrenaline anddopamine uptake blockers, dopamine I agonists, L-DOPA, nicotine andderivatives, and NO synthase modulators.

Growth Factors

Using various mechanisms, growth factors are compounds that promotesparticular cell to differentiate or proliferate. Some growth factorsinclude bone morphogenetic proteins (BMPs), epidermal growth factor(EGF), erythropoietin (EPO), fibroblast growth factor (FGF),granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophagecolony stimulating factor (GM-CSF), hepatocyte growth factors (HGF),insulin-like growth factor (IGF), myostatin (GDF-8), nerve growth factor(NGF), neurotrophins, platelet-derived growth factor (PDGF),thrombopoietin (TPO), Transforming growth factor alpha (TGF-α),Transforming growth factor beta (TGF-β), vascular endothelial growthfactor (VEGF), Interleukin-1 3 (IL-1β), IL-2, IL-3, IL-4, IL-6, IL-7,IL-8, IL-21, IL-33, M-CSF, Noggin, Interferons (IFN)-α, IFN-β and IFN-γ.

Compounds for Activating Potassium Channels TREK-1

Other suitable agents used in Western medicine include compounds capableof activating the potassium channels TREK-1. The activation of TREK-1has been found to diminish the effects of stroke or neurodegeneration inpredisposed subjects or subjects at risk of stroke or neurodegeneration.In addition, TREK-1 has been shown to play a role in treating patientswith diseased or injured tissues of the central or peripheral nervoussystem, and patients suffering from depression, psychiatric indications,natural aging and traumatic brain cell death. Since NeuroAid® does notactivate potassium channels TREK 1, compounds that are capable ofactivating the potassium channels TREK-1 may be used in combination withNeuroAid™ (MLC 601) to diminish the effects of stroke orneurodegeneration and treating patients with diseased or injured tissuesof the central or peripheral nervous system, and patients suffering fromdepression, psychiatric indications, natural aging and traumatic braincell death.

One example of a compound that is capable of activating the potassiumchannels TREK-1 is Polyunsaturated fatty acids (PUFAs). Polyunsaturatedfatty acids are fatty acids that contain more than one double bond.PUFAs can be categorized as methylene-Interrupted Polyenes or Conjugatedfatty acids.

Methylene-Interrupted Polyenes are fatty acids that have two or more cisdouble bonds that are separated from each other by a single methylenegroup. The essential fatty acids are all omega-3 and -6methylene-interrupted fatty acids. Examples of Omega-3 fatty acidsinclude without limitation: Alpha-linolenic acid (ALA), Stearidonic acid(STD), Eicosatrienoic acid (ETE), Eicosatetraenoic acid (ETA),Eicosapentaenoic acid (EPA), Docosapentaenoic acid (DPA),Docosahexaenoic acid (DHA), Clupanodonic acid, Tetracosapentaenoic acid,Tetracosahexaenoic acid or Nisinic acid. Examples of Omega-6 fatty acidsinclude without limitation: Linoleic acid (LIN), Gamma-linolenic acid(GLA), Eicosadienoic acid, Dihomo-gamma-linolenic acid (DGLA),Arachidonic acid (AA), Docosadienoic acid, Adrenic acid,Docosapentaenoic acid or Osbond acid. Omega-9 fatty acids are alsomethylene-Interrupted polyenes, and may be monosaturated orpolysaturated. Examples of Omega-9 fatty acids include withoutlimitation: Oleic acid, Eicosenoic acid, Mead acid, Erucic acid orNervonic acid.

Conjugated fatty acids are fatty acids that have two or more conjugateddouble bonds. Examples of Conjugated fatty acids include withoutlimitation: Remenic acid, α-Calendic acid, β-Calendic acid, Jacaricacid, α-Eleostearic acid, β-Eleostearic acid, Catalpic acid, Punicicacid, Rumelenic acid, α-Parinaric acid, β-Parinaric acid,Bosseopentaenoic acid.

Some other PUFAs which are not catergorized as methylene-InterruptedPolyenes or Conjugated fatty include without limitation: Pinolenic acidand podocarpic acid.

Other compounds that may be capable of activating the potassium channelsTREK-1 include the drug Riluzole (Rilutek®), Lysophospholids (LPLs),Caffeic Acid esters and Xenon. These compounds may also be used incombination with NeuroAid™ (MLC 601) to diminish the effects of strokeor neurodegeneration and treating patients with diseased or injuredtissues of the central or peripheral nervous system, and patientssuffering from depression, psychiatric indications, natural aging andtraumatic brain cell death.

Modes of Administration

NeuroAid™ (MLC 601) may be administered orally, parenterally,intravenously, subcutaneously, intradermally, intraperitoneally ortopically, in liquid, semi-liquid or solid form and are formulated in amanner suitable for each route of administration. When NeuroAid™ (MLC601) is administered orally, it may be administered as four 0.4 gcapsules being taken 3 times a day. For patients with swallowingdifficulties, capsules may be opened and powder diluted in water thatcan be drunk as such or injected via a gastric tube. Hence, a daily doseof about 4.8 g is envisaged. In one embodiment, the patient's daily doseof NeuroAid™ (MLC 601) is about 1 g to 8 g; 2 g to 8 g; 3 g to 7 g; 4 gto 6 g; 4.25 g to 5.75 g; 4.5 g to 5.25 g; 4.5 g to 5 g; 4.6 g to 4.10g; or 4.7 g to 4.9 g. A “daily dose” can be a single tablet or capsuleetc. or multiple tablets or capsules etc. to be taken on a given day.

In one embodiment, each course of NeuroAid™ (MLC 601) treatment lastsabout 4 weeks. Typically 3 courses are administered, most commonly backto back. No therapeutic window is required but additional courses can beadded even after a few days of treatment cessation. Hence, in oneembodiment, each NeuroAid™ (MLC 601) treatment lasts about 12 weeks. Inanother embodiment, the treatment course of NeuroAid™ (MLC 601) is about4 to 24 weeks; 7 to 16 weeks; 9 to 15 weeks; 10 to 14 weeks; or 11 to 13weeks.

In another embodiment, NeuroAid™ (MLC 601) can also be used as a chronictreatment to address chronic disease or as a preventive measure.

In one embodiment, NeuroAid™ (MLC 601) may be used as part of acombination therapy with western medicine that promotes cell growth. Thecombination partners of NeuroAid™ (MLC 601) and cell growth factor maybe present in a single formulation or may be present as separateformulations. In one embodiment there may be a synergistic effect.

The combination partners NeuroAid™ (MLC 601) and cell growth factor maybe administered to the patient at the same time (e.g. simultaneously) orat different times (e.g. sequentially) and over different periods oftime, which may be separate from one another or overlapping. Thecombination partners NeuroAid™ (MLC 601) and cell growth factor may beadministered in any order.

When cell growth factor is utilized and the appropriate administrationroute and dose level will be known to those in the art or could bereadily determined by one skilled in the art. Typically, as is wellknown in the medical art, dosage regimens may depend on various factorsincluding the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently. Whileindividual needs vary, determination of optimal ranges of effectiveamounts of each component is within the skill of the art. The dosagewould be similar to that administered when the agent is used withoutNeuroAid™ (MLC 601).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows bar charts of the dose-effect of NeuroAid™ (MLC 601)treatment on cell viability on day 8, day 10 and day 14. Threeconcentrations of NeuroAid™ (MLC 601) (0.1, 0.5, 1.0 μg/ml) werecompared to the control.

FIG. 2 shows bar charts of the effect of NeuroAid™ (MLC 601) treatmenton LDH release on day 10 and day 14. A concentration of 1.0 μg/mlNeuroAid™ (MLC 601) was compared to the control.

FIG. 3 shows confocal images of the effect of NeuroAid™ (MLC 601)treatment on neuronal DCX expression on day 3 and day 14. Aconcentration of 1.0 μg/ml NeuroAid™ (MLC 601) was compared to thecontrol.

FIG. 4 shows bar charts of the effect of NeuroAid™ (MLC 601) andNeuroAid II (MLC 901) treatments on cell viability on day 8, day 10, day12 and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0μg/ml NeuroAid II (MLC 901) were compared to the control.

FIG. 5 shows a line graph of the effect of NeuroAid™ (MLC 601) andNeuroAid II (MLC 901) treatments on LDH release on day 8, day 10, day 12and day 14. A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0μg/ml NeuroAid II (MLC 901) were compared to the control.

FIG. 6 shows confocal images of the effect of NeuroAid™ (MLC 601) andNeuroAid II (MLC 901) treatments on DCX expression on day 3 and day 14.A concentration of 1.0 μg/ml NeuroAid™ (MLC 601) and 1.0 μg/ml NeuroAidII (MLC 901) were compared to the control.

FIG. 7 shows schematic diagrams of the experimental protocols forNeuroAid II (MLC 901) treatments against stroke. FIG. 7A shows theexperimental protocol for Example 4, which investigates the effects ofNeuroAid II (MLC 901) treatment; FIG. 7B shows the experimental protocolfor Example 5, which investigates the effects of an acute NeuroAid II(MLC 901) treatment. Both studies assessed the rate of survival and alsoquantified the infarct volume at 24 h post-ischemia.

FIG. 8 shows a bar graph of the survival rate after NeuroAid II (MLC901) pre-treatment at 24 h post-ischemia.

FIG. 9 shows a bar graph of the survival rate (A) and infarct volume (B)after NeuroAid II (MLC 901) post-treatment at 24 h post-ischemia.

FIG. 10 shows bar charts on the comparative effects of NeuroAid II (MLC901), Rhizoma chuanxiong Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis treatments (1 μg/ml) on corticalneurons in culture estimated at Day 12 of treatment. FIG. 10A shows theeffect of the respective treatments on cell viability and FIG. 10B showsthe effect of the respective treatments on LDH release.

FIG. 11 shows bar charts on the comparative effects of a post-treatmentwith NeuroAid II (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensisand combined Rhizoma chuanxiong/Radix angelicae sinensis againstischemic brain injury in vivo. FIG. 11A and FIG. 11B shows the survivalrate (A) and infarct volume (B) respectively.

FIG. 12 shows bar charts on the comparative effects of a pre-treatmentwith NeuroAid II (MLC 901), Rhizoma chuanxiong and Radix angelicaesinensis against ischemic brain injury in vivo. FIG. 12A and FIG. 12Bshows the survival rate (A) and infarct volume (B) respectively.

FIG. 13 shows representative epifluorecence microscopy photographs ofthe comparative effects between NeuroAid II (MLC 901), Rhizomachuanxiong, Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis treatment (1 μg/ml) on in vitro DCXimmunoexpression in cultured cortical cells at Day 12 of treatment.

FIG. 14 shows bar charts of DCX signal intensity in immunostainedneurons observed in epifluorecence microscopy.

FIG. 15 shows bar charts of the neurite outgrowth obtained by measuringon epifluorecence microscopy, the total length of neurite WO in functionof treatments.

EXAMPLES

Non-limiting examples of the invention will be further described ingreater detail by reference to specific examples, which should not beconstrued as in any way limiting the scope of the invention.

Materials Medium for Cortical Sampling:

-   -   HBSS/I

NaCl 8 g/I

KCl 0.26 g/I

MgSO₄ 0.2 g/I

CaCl₂ 0.264 g/I

NaH₂CO₃ 2.24 g/I

NaH₂PO₄ 0.15 g/I in H₂O

-   -   HBSS+: add 6 g glucose

Cell Culture Medium

Neurobasal (21103-049; Invitrogen, San Diego, Calif.)

qB27 (n° 17504; Invitrogen, San Diego, Calif.)

Uridine (U3003; Sigma, St. Louis, Mo., USA)

Fluoro-deoxy-Uridine (46875; Fluka-Chemika-Biochemika, Buchs,Switzerland)

Glutamax (35050; Gibco-BRL Life Technologies. GmbH, Karlsrahe, Germany)

Antibiotics (Penicillin-streptomycin) (Gibco-BRL Life Technologies.GmbH, Karlsrahe, Germany)

Animals

All experiments were performed according to policies on the care and useof laboratory animals of European Community legislation. The localEthics Committee approved the experiments (protocol numbersNCA/2006/10-1 and NCA/2006/10-2). Adult male C57/B16 mice, weighing 22to 26 g were used in this study. The animals housed under controlledlaboratory conditions with a 12-hour dark-light cycle, a temperature of21±2° C., and a humidity of 60 to 70% for at least one week prior todrug treatment or surgery. The mice had free access to standard rodentdiet and tap water.

NeuroAid II (MLC 901) Drinking Solution

One capsule of NeuroAid II (MLC 901) was dissolved in 66 ml water understirring with an agitator for one hour at 37° C. The solution is thenfiltered with a 0.45 μm filter.

NeuroAid II (MLC 901) for Intraperitoneal Injections

30 mg from a NeuroAid II (MLC 901) capsule was diluted in 3 ml salinesolution corresponding to a concentration of 10 mg/ml (Stock solution)at 37° C. during 60 min. The concentration used in the experiment is 1μg/ml (injected volume: 500 μl). To obtain the dose of 1 μg/ml, thestock solution is diluted by 100. After dilution, the mixture isvortexed to obtain a good homogenization and filtered on a 0.45 μmfilter.

Methods A. Cortical Cell Culture

Time-pregnant (E14) C57B16/J mice were anesthetized with isopentanefollowed by cervical dislocation. Fetuses were removed and placed incold (Hanks Balanced Salt Solution) HBSS+solution. Cerebral corticeswere dissected in cold HBSS+solution and the meninges were removed. Thecortical samples were cut in small pieces and were gently trituratedwith a fire-polished glass Pasteur pipette in 8 nil HBSS+solution. Themix was filtered (40 μM filter) and centrifuged at 800 rpm for 8 min.The supernatant was removed and the pellet was dissolved in 2 ml culturemedium. Cells were plated on Polylysine-coated 12 well (24 mm diameter;Sigma-Aldrich Chimie, St. Quentin Fallavier, France)) plates with glasscoverslips (12 mm diameter; CML, Nemours, France) at a density of 1×10⁶cells/well. Cultures were maintained at 37° C. in a humidified 5% CO₂atmosphere incubator in Neurobasal supplemented with B27, Glutamax,antibiotics were used for experiments after 16 days. Glial growth wassuppressed by addition of 5-Fluoro-2-deoxyuridine (2 μM) and Uridine (2μM) during the second day of culture.

B. Neuroaid™ (MLC 601) Treatment

30 mg from a NeuroAid™ (MLC 601) capsule was diluted in 3 ml Neurobasalmedium corresponding to a concentration of 10 mg/ml (Stock solution) at37° C. for a duration of 60 minutes. The concentrations tested in theexperiments were: 1 μg/ml or 10 μg/ml of culture medium. 1 ml ofNeuroAid™ (MLC 601) solution was put in each 24 mm well of plate.

To obtain the dose of 1 μg/ml, the stock solution was diluted by 100times: 0.1 mg/ml (10 μl per 24 mm well/ml, corresponding to 1 μg/well).

To obtain the dose of 10 μg/ml, the stock solution was diluted by 10times: 1 mg/ml (10 μl per 24 mm well/ml, corresponding to 10 μg/well).

After dilution, the mixture was vortexed to obtain a good homogenizationand filtered on a 0.45 μm filter.

Cells were treated each day with NeuroAid™ (MLC 601), NeuroAid II (MLC901) or Neurobasal medium from Day 1 of culture.

C. Lactate Dehydrogenase (LDH) Measurements

Neuronal injury was quantitatively assessed by the measurement of lacticdehydrogenase (LDH) release from cultured neurons incubated in cellculture medium. At Day 1, 5, 8, 10, 12, 14 and 16, 100 μl of the cellculture medium was transferred from culture wells to 96-well plates andmixed with 100 μl of reaction solution according to LDH assay kit (RocheDiagnostic: Cytotoxicity Detection kit: ref 1644793, Indianapolis, USA).Optical density (OD) was measured 30 minutes later at 492 nm utilizing aLabsystem Multiscan microplate reader (Labsystem Multiscan RC, Finland).Background absorbance at 620 is subtracted. LDH activity is expressed asactivity present in the 25 μl medium volume. Results are expressed asOD×10⁻³.

D. Cell Viability

At Day 1, 5, 8, 10, 12, 14 and 16, the totality of cell culture mediumwas removed and replaced by 500 μl of Neurobasal medium+Cell Titer 96Aqueous One Solution kit: Cell Titer 96 (r) Aqueous One Solution CellProliferation Assay Kit. Neuronal viability was determined using theCell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay(Promega, Madison, USA). According to the Proliferation Assay Kitprotocol, cells are incubated for 4 hours at 37° C. in the humidified 5%CO₂ atmosphere incubator. The reaction was stopped with 2% SodiumDodecyl Sulfate (SDS). Optical density was measured 4 hours later at 490nm utilizing a Labsystem Multiscan microplate reader. Backgroundabsorbance at 620 was subtracted. Results were expressed as OD×10⁻³representating the number of viable cells.

Statistic analysis of cell viability and LDH results are assessed usingone factor (analysis of variance) ANOVA test followed by post-hoc test(P<0.05).

E. Double Cortin (DCX) Immunohistochemistry of Cortical Cell onCoverslips

Cortical cell are fixed onto coverslips with 4% paraformaldehyde inphosphate buffered saline (PBS), permeabilized in 0.3%polyoxyethylensorbitan monolaurate (Tween 20, Sigma) for 10 minutes andblocked with 2.5% donkey serum in PBS for 2 hours at room temperature.Coverslips were incubated with a goat anti-doublecortin (DCX) antibody(1:200, SC-8066, Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) in2% donkey serum/PBS overnight. After 3 washes in PBS, coverslips wereincubated in anti-goat Alexa 488-coupled antibodies (FluoProbes,Interchim, Montluçon, France) in 2% donkey serum for 2 hours, and washedthree times in PBS for 5 min each. The coverslips are then incubated inHoechst solution (3 μl in 10 ml; Sigma-Aldricht Chimie, Saint QuentinFallavier, France) for 10 minutes to label cell nuclei. After 2 washesin PBS and 1 wash in water, the coverslips are dried and mounted onglass slides with Fluoroprep (75521; BioMérieux, Marcy l′Etoile,France). Cells were observed using epifluorescence and confocalmicroscopy. Confocal microscopy observations are performed using a LaserScanning Confocal Microscope (TCS SP, Leica Microsystems HeidelbergGmbH, based in Mannheim, Germany) equipped with a DMIRBE invertedmicroscope and an argon-krypton laser (laser excitation 488 nm,acquisition 500-600 nm every 10 nm). Signal specificity was assessed innegative control coverslips by omitting primary antibody directedagainst the DCX protein. Images were acquired as single transcellularoptical sections and averaged over at least four scans per frame.Confocal images of DCX-Alexa-488 antibody labeling were then obtainedafter spectral correction of the autofluorescence background.

F. Physiological Parameters Used when Investigating the Effects of aNeuroAid II (MLC 901) in C57BI/6 Mice

General anesthesia was induced with 3% isoflurane and maintained with 1%isoflurane by means of an open facemask for each mouse. Mice wereallowed to breathe spontaneously. A subset of animals (n=5 per group)were monitored for physiological parameters including mean arterialblood pressure (MABP), rectal temperature, arterial blood gases and pHbefore, during and after ischemia. The right femoral artery wascatheterized with PE-10 polyethylene tubing and connected to a bloodpressure transducer (Harvard Apparatus, Massachusetts, USA) forcontinuous monitoring of MABP (mm Hg). A heparinized blood sample (751.11) was then obtained from the catheterized femoral artery and bloodpO₂, pCO₂ and pH were measured using an Acid-Base Laboratory system (ABL555, Radiometer). Core temperature was monitored continuously with athermometer (3-mm probe diameter; Harvard Apparatus, Massachusetts,USA), inserted into the rectum and maintained at physiologicaltemperatures using a thermostatically controlled heating blanket(Harvard Apparatus, Massachusetts, USA). Core temperature was maintainedat physiological values by a combination of the homeothermic blanketcontrol.

G. Induction of Transient Focal Cerebral Ischemia in C57131/6 Mice

Focal ischemia was induced by occlusion of the left middle cerebralartery (MCA) using an intraluminal filament technique (Heurteaux et al,2006) After a midline neck incision was made, the left common andexternal carotid arteries were isolated and ligated with a silk 4-0 silksuture (Ethicon). A temporary yasargil aneurysm clip (BMH31, Aesculap,Tuttlingen, Germany) was temporarily placed on the internal carotidartery. A 6-0 coated filament (Doccol, Redlands, Calif., USA), bluntedat tip with an open flame, was introduced through a small incision intothe common carotid artery and 13 mm distal to the carotid bifurcationfor occlusion of the origin of the MCA. Animals were kept at 37° C. forone hour, after which time the thread was carefully withdrawn to allowreperfusion of the MCA territory. To control the MCAO severity regionalCBF (rCBF) was determined by laser-Doppler flowmetry (Perimed) using aflexible 0.5-mm fiber optic extension to the masterprobe fixed on theintact skull over the ischemic cortex (2 mm posterior and 6 mm lateralfrom the bregma). Sham-operation was performed inserting the thread intothe common carotid artery without advancing it to occlude the MCA. Theanimals were allowed to regain full consciousness on a heating padbefore returning to the cage.

H. Determination of Infarct Volume

To assess the infarct volume in the acute NeuroAid II (MLC 901)post-treatment study (Example 5), mice were sacrificed at 24 hours afterreperfusion. Their brains were removed and sectioned into six 1 mm-thickcoronal slices using a tissue chopper (Phymep, France). Coronal brainslices were immediately immersed into 2% 2, 3, 5-Triphenyltetrazoliumchloride (TTC, Sigma, France) for 20 min at room temperature in the darkfollowed by fixation in a 4% paraformaldehyde solution overnight priorto analysis as described previously (Ding-Zhou et al., 2002). Thestriatal and cortical areas of infarction, outlined in light weremeasured on each section using a computer image analysis system andcorrected for brain edema according to Golanov and Reis (Golanov andReis, 1995). Infarct volume, expressed in mm³ was calculated by a linearintegration of the corrected lesions areas.

In addition cresyl violet, a dye that stains the Nissl bodies in thestellate somas of viable neurons was used to confirm the evolution ofinfarct volume in ischemic mice. Coronal frozen sections of brain (10μm-thick) were added to a solution of 1% cresyl violet in 0.25% aceticacid for 3 min, rinsed, dehydrated and mounted with Entellan. Sectionswere analyzed under light microscopy.

Example 1 Dose-Effect of NeuroAid™ (MLC 601) on Cell Viability and LDHRelease

Cortical cells, as prepared by the methods disclosed above, were exposedto four concentrations of NeuroAid™ (MLC 601): 0.1, 0.5, 1.0, 10 μg/mlfrom Day 1 until Day 14 of culture. Cell viability was studied at Day 8,10 and 14 by the methods disclosed above and the results are shown inFIG. 1.

The effect of NeuroAid™ (MLC 601) treatment against neurodegeneration ofcortical cells over time in culture was also analyzed by LDH release.Increasing cell suffering that leads to cell death is associated withincreased LDH release. LDH release was measured on day 10 and day 14 bythe methods disclosed above and the results are shown in FIG. 2.

Results

FIG. 1 shows that at Day 14, a NeuroAid™ (MLC 601) treatment at aconcentration of 1 μg/ml induced a significant increase (35%) inneuronal survival as compared to control (**P<0.01). Until Day 10 therewas no significant difference in the percentage of cell viability oncells treated with NeuroAid™ (MLC 601) concentrations of 0.1-0.5-1.0μg/ml as compared to control (P>0.05) (n=10 wells per experimentalgroup).

FIG. 2 shows the ratio of LDH release/cell viability at Day 10 and 14 atthe concentrations of 1 μg/ml NeuroAid™ (MLC 601). Compared to control,NeuroAid™ (MLC 601) treatment significantly reduced the LDH releaseafter 10 and 14 days of culture (P<0.01) (n=10 wells per experimentalgroup).

Based on the experimental data in Example 1, it can be demonstrated thattreatment with NeuroAid™ (MLC 601) in cortical cells in culturedemonstrates an increase of cell viability and a reduction of LDHrelease, which is a marker of the cell suffering. Example 1 alsodemonstrates that NeuroAid™ (MLC 601) may be used for diminishing theeffect of stroke or neurodegeneration in predisposed subjects orsubjects at risk of stroke or neurodegeneration, for treating patientswith diseased or injured tissues of the central or peripheral nervoussystems, or for use as a cell culture reagent.

Example 2 Effect of NeuroAid™ (MLC 601) on NeuronalProliferation/Neurogenesis

DCX is a highly hydrophilic microtubule-associated protein that wasexpressed specifically in all migrating precursors of the developing CNSand in areas of continuous neurogenesis in adult brain. Neuronalproliferation was analyzed from DCX expression in cortical cells inculture by the methods disclosed above. Cortical cells, as prepared bythe methods disclosed above, were treated with 1 μg/ml NeuroAid™ (MLC601) from Day 1 until Day 14 of culture. The cells were then preparedand observed using epifluorescence and confocal microscopy as describedin the methods above to determine DCX expression. The results are shownin FIG. 3.

Results

In FIG. 3, staining with an antibody against DCX shows that at Day 3,there was no difference in DCX expression between the control andcortical cells treated with 1 μg/ml NeuroAid™ (MLC 601). However, at Day14, DCX immunoreactivity disappeared in the control, while there was anincrease of DCX expression induced by NeuroAid™ (MLC 601) treatment,highlighting the development of an important axonal and dendriticnetwork.

Because of its association with neurogenic processes, the DCX protein iscurrently used as a marker for neurogenesis. DCX is amicrotubule-associated protein whose expression is associated with allmigrating neuronal precursors in fetal and adult brain. DCX appears tobe important for the normal developmental migration of cortical neurons,because mutations in DCX in humans lead to syndromes characterized bymigrational arrest of these neurons and which is manifested clinicallyby subcortical laminar heterotopias, mental retardation and seizures.DCX is also expressed in some mature neurons in the adult brain, whereit is involved in axonal outgrowth and synaptogenesis.

Thus, based on the experimental data of Example 2, it can bedemonstrated that treatment with NeuroAid™ (MLC 601) results in anincrease in DCX expression in cortical cells, suggesting strongly thattreatment with NeuroAid™ (MLC 601) enhances neuroproliferation,neurogenesis, and neurorepair necessary to restore neurological function(such as motor and cognitive). Example 2 also demonstrates thatNeuroAid™ (MLC 601) may be used for promoting neuronal outgrowth andproliferation of neurons in tissues of the central or peripheral nervoussystems, for promoting cell growth, and for a method for inducing cellsurvival, growth, proliferation or differentiation of cells usingNeuroAid™ (MLC 601).

Example 3 Effect of NeuroAid™ (MLC 601) and NeuroAid II (MLC 901)Treatments on Cell Viability, LDH Release and Neuronal Proliferation

Cortical cells were exposed to a concentration of 1 μg/ml NeuroAid™ (MLC601) or NeuroAid II (MLC 901) from Day 1 until Day 14 of culture. Thisconcentration of 1 μg/ml corresponds to the best results obtained oncell viability and LDH release as described above. Cell viability wasstudied at Day 8, 10, 12 and 14. Neuronal proliferation in the course oftime was analyzed by observing DCX expression in cortical cells inculture treated with 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC901).

Results

FIG. 4 shows that at Day 8, a significant increase in neuronal viabilityas compared to respective control (**P<0.01; ***P<0.001) was observed incortical cells treated with 1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II(MLC 901). However, the highest efficiency of both treatment wasobserved at Day 14 with 45% increase of cell survival (***P<0.001).There was no significant difference of efficiency between NeuroAid™ (MLC601) and NeuroAid II (MLC 901) at the different stages of culture (n=10wells per experimental group).

FIG. 5 shows the comparison of NeuroAid™ (MLC 601) and NeuroAid II (MLC901) treatments on the ratio of LDH release/cell viability at Day 8, 10,12 and 14 at the concentrations of 1 μg/ml. Compared to the control,both treatments significantly reduced LDH release after 12 and 14 daysof culture (*P<0.05 and ***P<0.001) (n=10 wells per experimental group).There was no significant difference of efficiency on LDH release betweenNeuroAid™ (MLC 601) and NeuroAid II (MLC 901) treatments.

FIG. 6 shows that, compared to the control, cortical cells treated with1 μg/ml NeuroAid™ (MLC 601) or NeuroAid II (MLC 901) developed a muchdenser axonal and dendritic network from Day 3 in culture. At Day 14,DCX immunoreactivity disappeared in Control and the labeling of the DCXprotein became different between cortical cells treated with NeuroAid™(MLC 601) and cortical cells treated with NeuroAid II (MLC 901). The DCXprotein was always expressed in processes of cortical cells treated byNeuroAid™ (MLC 601) or NeuroAid II (MLC 901). However, it appeared thatin cortical cells treated with NeuroAid II (MLC 901), there was also astrong increase of DCX immunoreactivity in the cytoplasm of these cells.

The results obtained with both NeuroAid™ (MLC 601) treatments (NeuroAid™(MLC 601) and NeuroAid II (MLC 901)) show an increase in cell survival,an increase in LDH release and an increase of DCX expression in corticalcells. There was no significant difference between the results ofNeuroAid™ (MLC 601) and NeuroAid II (MLC 901). Thus, based on theexperimental data, it can be demonstrated that both NeuroAid™ (MLC 601)treatments (NeuroAid™ (MLC 601) and NeuroAid II (MLC 901)) enhanceneuroproliferation, neurogenesis and neurorepair and may be a form ofenhanced treatment for neurological diseases and nervous systeminjuries.

The experimental results above also support the use of extracts ofNeuroAid™ (MLC 601) and NeuroAid II (MLC 901) for improvement ofconnectivity and survival of all types of neuronal cultures. Theseextracts could be systematically added to classical culture media forneuronal cultures to considerably improve viability, dendritic growthand synaptogenesis. Example 3 also demonstrates that (NeuroAid™ (MLC601) and NeuroAid II (MLC 901) may be used for promoting neuronaloutgrowth and proliferation of neurons in tissues of the central orperipheral nervous systems, for promoting cell growth, for treatingpatients having a condition selected from the group of depression,psychiatric indications, natural aging and traumatic brain cell death,diminishing the effect of stroke or neurodegeneration in predisposedsubjects or subjects at risk of stroke or neurodegeneration, promotincell growth, for treating patients with diseased or injured tissues ofthe central or peripheral nervous systems, for use as a cell culturereagent and for a method for inducing cell survival, growth,proliferation or differentiation of cells.

Example 4 The Effect of NeuroAid II (MLC 901) Pretreatment in Mice 24 hPost-Ischemia

In this study, mice (n=11) were pretreated for six weeks with a drinkingsolution of NeuroAid II (MLC 901) and the rate of survival was analyzedat 24 h post-ischemia (FIG. 7A). Control mice (n=10) received a tapwater drinking solution.

To determine whether a pretreatment of NeuroAid II (MLC 901) increasesthe rate of survival of mice subjected to ischemia, the animals weretreated with NeuroAid II (MLC 901) during six weeks before the inductionof ischemia. FIG. 8 shows that a six week-pretreatment of NeuroAid II(MLC 901), given in the drinking induced a strong reduction of themortality of NeuroAid II (MLC 901)-treated animals, compared to controlmice. The NeuroAid II (MLC 901) pretreatment during six weeks induced asurvival rate of 90.9% as compared to 40.0% in control group.

Example 4 demonstrates that NeuroAid is beneficial before an ischemicstroke. These studies illustrates that NeuroAid advantageously reducedthe infarct size and mortality rate of ischemic mice in a clinicallyrelevant model of stroke.

Example 5 The Effect of Acute NeuroAid II (MLC 901) Post-Treatment inMice 24 h Post-Ischemia

In this study, mice (n=10) received an acute post-treatmentintraperitoneal injection of NeuroAid II (MLC 901), given at the onsetof ischemia and 6 h after reperfusion. Control mice (n=10) was injectedwith saline solution. The survival rate and the infarct volumes werequantified at 24 h of reperfusion.

To determine whether acute administration of NeuroAid II (MLC 901)protects against ischemic stroke, mice (n=10) were subjected to ischemiaand injected intraperitonally with a single dose of 1 mg/ml of NeuroAidII (MLC 901) solution at the onset of ischemia and 6 h afterreperfusion. Acute administration of NeuroAid II (MLC 901) induced asurvival rate of 90.0% compared to 38.8% observed in control mice (FIG.9A). This NeuroAid II (MLC 901) post-treatment drastically decreasedcerebral infarction as shown in FIG. 9B. The NeuroAid post-treatmentreduced the stroke volume by 47.4% (P<0.001) as compared to controlmice.

These studies demonstrate that NeuroAid is beneficial both before andafter an ischemic stroke. These studies illustrates that NeuroAidadvantageously reduced the infarct size and mortality rate of ischemicmice in a clinically relevant model of stroke.

Methods for Examples 6 to 8 A. Cortical Cell Culture

Time-pregnant (E14) C57BI6/J mice were anesthetized with isopentanefollowed by cervical dislocation. Fetuses were removed and placed incold (Hanks Balanced Salt Solution) HBSS+solution. Cerebral corticeswere dissected in cold HBSS+solution and the meninges were removed. Thecortical samples were cut in small pieces and were gently trituratedwith a fire-polished glass Pasteur pipette in 8 nil HBSS+solution. Themix was filtered (40 μM filter) and centrifuged at 800 rpm for 8 min.The supernatant was removed and the pellet was dissolved in 2 ml culturemedium. Cells were plated on Polylysine-coated 12 well (24 mm diameter;Sigma-Aldrich Chimie, St. Quentin Fallavier, France)) plates with glasscoverslips (12 mm diameter; CML, Nemours, France) at a density of 1×10⁶cells/well. Cultures were maintained at 37° C. in a humidified 5% CO₂atmosphere incubator in Neurobasal supplemented with B27, Glutamax,antibiotics were used for experiments after 16 days. Glial growth wassuppressed by addition of 5-Fluoro-2-deoxyuridine (2 μM) and Uridine (2μM) during the second day of culture.

Experiments were monitored by one researcher blinded to the treatmentstatus (n=3 cultures, 36 wells per experimental group).

B. Cell Injury Assay: Cell Survival and Lactate Dehydrogenase (LDH)Measurements

Cell viability was assessed at Day 12 of cell culture by using the CellTiter 96 (r) Aqueous One Solution Cell Proliferation Assay (Promega,Charbonnières-les-Bains, France) (n=3 cultures, 36 wells perexperimental group). The assay was a colorimetric method, which wasbased on the use of the3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliuminner salt (MTS), a marker of mitochondrial activity and anelectron-coupling reagent (phenazine ethosulfate, PES). The MTStetrazolium compound was bioreduced by cells into a colored formazanproduct that is soluble in tissue culture medium.

At Day 12, the totality of cell culture medium was removed and replacedby 500 μl of Neurobasal medium+Cell Titer 96 Aqueous One Solution kit:Cell Titer 96 (r) Aqueous One Solution Cell Proliferation Assay Kit.According to the Proliferation Assay Kit protocol, cells were incubatedfor 4 hours at 37° C. in the humidified 5% CO2 atmosphere incubator. Thereaction was stopped with 2% Sodium Dodecyl Sulfate (SDS). Opticaldensity was measured 4 hours later at 490 nm utilizing a LabsystemMultiscan microplate reader (Labsystem Multiscan RC, VWR International,Fontenay sous Bois, France). Background absorbance at 620 wassubtracted. Results were expressed in Optical Density (OD×10-3)representating the number of viable cells. Data was expressed as thepercentage of cell viability, which was calculated by dividing theabsorbance value of treated samples by that of the untreated controlwithin each group.

Neuronal injury was quantitatively assessed by the measurement of lacticdehydrogenase (LDH) release from cultured neurons incubated in cellculture mediu at Day 12 of cell culture (Koh and Choi, 1987). The LDHrelease assay provided a measure of cytoplasmic membrane integrity. AtDay 12, 100 μl of the cell culture medium was transferred from culturewells to 96-well plates and mixed with 100 μl of reaction solutionaccording to LDH assay kit (Roche Diagnostic: Cytotoxicity Detectionkit: ref 1644793, Indianapolis, USA). Optical density (OD) was measured30 minutes later at 492 nm utilizing a Labsystem Multiscan microplatereader (Labsystem Multiscan RC, VWR International, Fontenay sous Bois,France). Background absorbance at 620 is subtracted. Neurons exposed toa lysis solution (PBS containing 0.1% Triton X-100) were used aspositive control and set as 100% LDH release. Data was expressed as theratio of LDH efflux/cell viability.

Results corresponded to the mean of three independent experiments withtriplicate determination. Statistical analyses of cell viability and LDHresults were assessed using one factor (analysis of variance) ANOVA testfollowing by post-hoc test (P<0.05).

C. Focal Ischemia

The researchers, who carried out the ischemic surgery and measuredinfarct volumes were double-blinded in regard to the treatment code.

Model of Focal Ischmia

Focal ischemia was induced on Adult male C57/B16 mice, weighing 22 to 26g (7-9 weeks old) by occlusion of the left middle cerebral artery (MCA)using an intraluminal filament technique (Huang et al., 1994) The leftcommon and external carotid arteries were isolated and ligated with asilk 4-0 silk suture (Ethicon). A temporary yasargil aneurysm clip(BMH31, Aesculap, Tuttlingen, Germany) was temporarily placed on theinternal carotid artery. A 6-0 coated filament (Doccol, Redlands,Calif., USA), was introduced through a small incision into the commoncarotid artery and 13 mm distal to the carotid bifurcation for occlusionof the origin of the MCA. Animals were kept at 37° C. for one hour,after which time, the thread was carefully withdrawn to allowreperfusion of the MCA territory. To control the MCAO severity regionalCBF (rCBF) was determined by laser-Doppler flowmetry (Perimed) using aflexible 0.5-mm fiber optic extension to the masterprobe fixed on theintact skull over the ischemic cortex (2 mm posterior and 6 mm lateralfrom the bregma). Sham-operation was performed inserting the thread intothe common carotid artery without advancing it to occlude the MCA. Theanimals were allowed to regain full consciousness on a heating padbefore returning to the cage.

Determination of Infarct Volume

At 30 hours after reperfusion, cresyl violet staining on coronal frozenbrain sections (10 μm-thick) was performed using a solution of 1% cresylviolet in 0.25% acetic acid and mounted with Entellan. The striatal andcortical areas of infarction, outlined in light were measured on eachsection using a computer image analysis system and corrected for brainedema according to Golanov and Reis (Golanov and Reis, 1995). Infarctvolume, expressed in mm³ was calculated by a linear integration of thecorrected lesions areas as previously described (Heurteaux et al.,2006a).

D. Drug Treatments

The composition of NeuroAid II (MLC 901) used (0.4 g per capsule) was asfollows:

0.57 g Radix astragali,

0.114 g Radix salvia miltiorrhizae,

0.114 g Radix paeoniae rubra,

0.114 g Rhizoma chuanxiong,

0.114 g Radix angelicae sinensis,

0.114 g Carthamus tinctorius,

0.114 g Prunus persica,

0.114 g Radix polygalae,

0.114 g Rhizoma acori tatarinowii,

0.0665 Hirudo.

For in vitro experiments, cell treatment with NeuroAid II (MLC 901),Rhizoma chuanxiong, Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis were at the concentration of 1μg/ml, starting at Day 3 of culture during 12 days (corresponding to 15days of culture).

For in vivo post-treatment, mice were intraperitonally injected with asingle dose of 2 μg/ml NeuroAid II (MLC 901) solution diluted in saline(as vehicle) in a total volume of 500 μl/mouse weighing 25 g, 3 and 24hours following the end of ischemia. For in vivo pre-treatment, NeuroAidII (MLC 901), Rhizoma chuanxiong, Radix angelicae sinensis and combinedRhizoma chuanxiong/Radix angelicae sinensis pre-treatment was given indrinking water at the concentration of 6 mg/ml during the 6 weeks beforethe induction of ischemia.

E. Immunohistochemistry on Cortical Neurons in Culture

Cortical cells were fixed on coverslips with 4% paraformaldehyde/PBS,permeabilized in 0.3% polyoxyethylensorbitan monolaurate (Tween 20,Sigma) for 10 min and blocked with 2.5% donkey serum in PBS for 2 hoursat room temperature. Cells were incubated with a goat anti-doublecortin(DCX) antibody (1:200, SC-8066, Santa Cruz Biotechnology Inc., SantaCruz, Calif.) in 2% donkey serum/phosphate buffer saline overnight(Heurteaux et al., 2006b). After 3 washes in phosphate buffer saline(PBS), cells were incubated in anti-goat Alexa 488-coupled antibodies(FluoProbes, Interchim, Montlucon, France) in 2% donkey serum for 2hours and washed three times in PBS for 5 minutes each. The neurons werethen incubated in Hoechst solution (3 μl in 10 ml, Sigma-AldrichtChimie, Saint Quentin Fallavier, France) for 10 min to label cellnuclei. After 2 washes in PBS and 1 wash in water, the coverslips weredried and mounted on glass slides with Fluoroprep (75521; Biomérieux,Marcy l′Etoile, France. Cells were observed using epifluorescencemicroscopy.

Signal specificity was assessed in negative control coverslips byomitting primary antibody. Epifluorescence microscopy images of proteinlabeling were captured with identical time of exposition after spectralcorrection of the autofluorescence background. The differentiatedneurites of cortical neurons in culture were observed by DCXimmunostaining at Day 12 of treatment. Neurite outgrowth was determinedon epifluorescence microscopy by measuring total length of neurites inculture dishes at different times of treatment using a cell photo imageand Neurite Tracer Image J software (Pool et al., 2008).

F. Statistical Analyses

Data was expressed as mean±S.E.M. Statistical analysis of differencesbetween groups was performed by using unpaired t test or ANOVA. Where Fratios were significant, statistical analyses were extended and post-hoccomparisons made by using Tukey's test multiple comparison tests. In allanalyses, the level of significance was set at P<0.05.

Example 6 Comparative Effects Between NeuroAid II (MLC 901), RhizomaChuanxionq, Radix Angelicae Sinensis and Combined RhizomaChuanxionq/Radix Angelicae Sinensis on Cell Viability and LDH Release

Cortical cells, as prepared by the methods disclosed above, were exposedto a concentration of 1 μg/ml NeuroAid II (MLC 901), Rhizoma chuanxiong,Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicaesinensis during 12 days.

The effects of NeuroAid II (MLC 901) to those of Rhizoma chuanxiong,Radix angelicae sinensis or combined Rhizoma chuanxiong/Radix angelicaesinensis treatments against neurodegeneration of cortical cells werecompared at Day 15 of culture (Day 12 of treatment) by using cellviability and LDH measurements as disclosed in the methods above. Theresults are shown in FIG. 10.

Results

FIG. 10A shows that at the concentration of 1 μg/ml, the threetreatments induced a significant increase in neuronal viability ascompared to control (*P<0.05, **P<0.01, ***P<0.001) However, NeuroAid II(MLC 901) demonstrated a higher efficacy on cell survival compared toRhizoma chuanxiong or Radix angelicae sinensis treatments. As shown inFIG. 10A, a NeuroAid II (MLC 901) treatment induced an approximately 51%increase of cell viability, as compared to the control (***P<0.001),while the increase in cell survival induced by Rhizoma chuanxiong, Radixangelicae sinensis and combined Rhizoma chuanxiong/Radix angelicaesinensis was of 37%, 29.5% and 35% respectively. There was a significantdifference of efficacy between NeuroAid II (MLC 901) and Rhizomachuanxiong, Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis treatments (##P<0.1) (n=36 wells pergroup)

It is well known that increased cell suffering that leads to cell deathis associated with increased Lactate DeHydrogenase (LDH) release. FIG.10B shows that compared to the control, the three treatmentssignificantly reduced the ratio LDH release/cell viability after 12 daysof treatment (*P<0.05 and **P<0.01). FIG. 10B also shows that NeuroAidII (MLC 901) significantly reduced LDH release when compared to Rhizomachuanxiong, Radix angelicae sinensis treatment or combined Rhizomachuanxiong/Radix angelicae sinensis (##P<0.01) (n=36 wells per group).

Based on the experimental data in Example 6, it can be demonstrated thatNeuroAid II (MLC 901) is more potent in increasing cell viability andreducing LDH release, in culture than either Rhizoma chuanxiong, Radixangelicae sinensis or combined Rhizoma chuanxiong/Radix angelicaesinensis.

Example 7

Comparative in vivo effects of NeuroAid II (MLC 901), Rhizomachuanxiong, Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis against ischemic brain injury invivo

Post-Treatment: To compare the effects of NeuroAid II (MLC 901), Rhizomachuanxiong, Radix angelicae sinensis and combined Rhizomachuanxiong/Radix angelicae sinensis in vivo, each treatment was testedin a mouse model of focal ischemia.

Ischemia was induced by transient middle cerebral artery occlusion(MCAO) for 60 min (Huang et al., 1994). Mice (n=12) were subjected tofocal ischemia and intraperitoneally injected at 3 hours and then againat 24 hours after MCAO with a single dose of 1 μg of NeuroAid II (MLC901), Rhizoma chuanxiong, Radix angelicae sinensis or combined Rhizomachuanxiong/Radix angelicae sinensis solution.

Acute administration of each treatment induced a significant survivalrate, with a better efficiency obtained with NeuroAid II (MLC 901). FIG.11A shows that a survival rate of 89% was achieved with NeuroAid II (MLC901) as compared to a survival rate of 58.5%, 64%, 73% and 75% inischemic vehicle-, rhizoma chuanxiong-, radix angelicae-treated andcombined Rhizoma chuanxiong/Radix angelicae sinensis mice, respectively.

The drastic decrease of cerebral infarction as shown in FIG. 11Bconfirms that the best result was observed with NeuroAid II (MLC 901).FIG. 11B shows that NeuroAid II (MLC 901) reduced the stroke volume by48.4% (′P<0.001) as compared to control ischemic mice at 30 hpost-ischemia. The infarct size in rhizoma chuanxiong-, radixangelicae-treated and combined Rhizoma chuanxiong/Radix angelicaesinensis mice was significantly larger than in NeuroAid II (MLC901)-treated animals after 30 hours of reperfusion (FIG. 11B, #P<0.05,##P<0.001 versus NeuroAid II (MLC 901) group).

Pre-treatment: To compare the potential in vivo effects between MLC 901,Rhizoma chuanxiong and Radix angelicae sinensis in prevention againststroke, each treatment was tested in pre-treatment.

The animals (n=12) were treated with NeuroAid II (MLC 901), Rhizomachuanxiong or Radix angelicae sinensis administered in the drinkingwater (6 mg/ml) for 6 weeks before the induction of ischemia. There wasno significant difference in the consumption of food and drinkingsolution between vehicle and NeuroAid II (MLC 901) treated groups. FIG.12A shows that a 6 week pretreatment of NeuroAid II (MLC 901), Rhizomachuanxiong and Radix angelicae sinensis induced a reduction of themortality of treated animals, compared to the control ischemic mice.

Pre-treatment induced a survival rate of 92%, 78% and 70% in NeuroAid II(MLC 901), Rhizoma chuanxiong and Radix angelicae sinensis, respectivelywhen compared to 70.5% in the control group. The best result fordiminishing the effects of stroke or neurodegeneration in predisposedsubjects or subjects at the risk of stroke or neurodegeneration obtainedwith. NeuroAid II (MLC 901) was confirmed by a significant decrease ofthe infarct volume, which was 39% and 24.5% less important as comparedto Rhizoma chuanxiong and Radix angelicae sinensis (FIG. 3B, P<0.01).

These studies in Example 7 demonstrate that NeuroAid II (MLC 901) ismore potent than either Rhizoma chuanxiong or Radix angelicae sinensisin pre-treatment of focal ischemia and NeuroAid II (MLC 901) is morepotent than either Rhizoma chuanxiong, Radix angelicae sinensis orcombined Rhizoma chuanxiong/Radix angelicae sinensis in post-treatmentof focal ischemia.

Example 8 Comparative Effects of NeuroAid II (MLC 901), RhizomaChuanxiong, Radix Angelicae Sinensis and Combined RhizomaChuanxiong/Radix Angelicae Sinensis on Neuroproliferation and NeuriteOutgrowth

To analyze the effects of NeuroAid II (MLC 901), Rhizoma chuanxiong,Radix angelicae sinensis and combined Rhizoma chuanxiong/Radix angelicaesinensis on neuronal proliferation and neurite outgrowth, the expressionof DCX in cultured cortical cells from embryonic mice was compared after14 days of treatment.

Representative epifluorescence microscopy images of DCX staining areshown in FIG. 13. FIG. 13 shows that there was an increase of DCXexpression induced by NeuroAid II (MLC 901), Radix angelicae sinensistreatment or combined Rhizoma chuanxiong/Radix angelicae sinensis,highlighting the development of an important axonal and dendriticnetwork with these three treatments as compared to cells treated withthe vehicle group or Rhizoma chuanxiong alone.

Quantification of the fluorescence intensity in each epifluorescencemicroscopy image was shown in FIG. 14. FIG. 14 showed that the bestneuroproliferative effect was obtained in cortical cultures treated withNeuroAid II (MLC 901).

To investigate whether NeuroAid II (MLC 901), Rhizoma chuanxiong, Radixangelicae sinensis or combined Rhizoma chuanxiong/Radix angelicaesinensis treatment could promote neurite outgrowth, the total length ofneurites in cultured cortical neurons was measured at Day 14 oftreatment.

FIG. 15 shows that NeuroAid II (MLC 901), Radix angelicae sinensis andcombined Rhizoma chuanxiong/Radix angelicae sinensis induced asignificant neurite outgrowth when compared to vehicle group (*P<0.05,***P<0.001). The best neurite outgrowth promoting activity was observedfor NeuroAid II (MLC 901) when compared to that of Radix angelicaesinensis alone or combined Rhizoma chuanxiong/Radix angelicae sinensis(#P<0.05).

These studies in Example 8 demonstrate that NeuroAid II (MLC 901) ismore potent than either Rhizoma chuanxiong, Radix angelicae sinensis orcombined Rhizoma chuanxiong/Radix angelicae sinensis for producingneuroproliferation and neurite outgrowth.

Applications

Advantageously, the disclosed compositions and methods provide a newtherapeutic strategy, which focuses on neural repair and restoringneurological functions.

The disclosed methods provide regenerative therapies for treatingnervous system injuries and neurological diseases, by promoting neuralgrowth so as to enable damaged or diseased nerves to function again.

The disclosed methods can also be used to promote in vitro cell growth.Advantageously, the in vitro culture of various cells may be used fortissue engineering or ex vivo therapeutic uses.

It will be apparent that various other modifications and adaptations ofthe invention will be apparent to the person skilled in the art afterreading the foregoing disclosure without departing from the spirit andscope of the invention and it is intended that all such modificationsand adaptations come within the scope of the appended claims.

1.-73. (canceled)
 74. A composition comprising the following four herbalcomponents: i) Radix Astragali; ii) Radix Polygalae; iii) RhizomaChuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and ivare present in the composition in a ratio of about 5:1:1:1 by weight,respectively.
 75. A composition consisting essentially of the followingfour herbal components: i) Radix Astragali; ii) Radix Polygalae; iii)Rhizoma Chuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii,iii, and iv are present in the composition in a ratio of about 5:1:1:1by weight, respectively.
 76. An extraction product of a compositioncomprising the following four herbal components: i) Radix Astragali; ii)Radix Polygalae; iii) Rhizoma Chuanxiong; and iv) Radix Angelicaesinensis, wherein i, ii, iii, and iv are present in the composition in aratio of about 5:1:1:1 by weight, respectively.
 77. An extractionproduct of a composition consisting essentially of the following fourherbal components: i) Radix Astragali; ii) Radix Polygalae; iii) RhizomaChuanxiong; and iv) Radix Angelicae sinensis, wherein i, ii, iii, and ivare present in the composition in a ratio of about 5:1:1:1 by weight,respectively.
 78. The composition according to claim 74, wherein saidcomposition is a pharmaceutical composition comprising apharmaceutically acceptable additive, carrier or diluent.
 79. Thecomposition according to claim 75, wherein said composition is apharmaceutical composition comprising a pharmaceutically acceptableadditive, carrier or diluent.
 80. The product according to claim 76,wherein said product is a pharmaceutical product comprising apharmaceutically acceptable additive, carrier or diluent.
 81. Theproduct according to claim 77, wherein said product is a pharmaceuticalproduct comprising a pharmaceutically acceptable additive, carrier ordiluent.
 82. The product according to claim 80, wherein saidpharmaceutical product is administered orally.
 83. The product accordingto claim 81, wherein said pharmaceutical product is administered orally.84. The product according to claim 80, wherein said pharmaceuticalproduct is administered orally as one or more tablets or capsules. 85.The product according to claim 81, wherein said pharmaceutical productis administered orally as one or more tablets or capsules.
 86. Theproduct according to claim 80, wherein said pharmaceutical product isused in combination with one or more Western medicines.
 87. The productaccording to claim 81, wherein said pharmaceutical product is used incombination with one or more Western medicines.